Current methods for assessing risk for or diagnosing diseases often rely on a diagnosis by attrition, a process of elimination or by invasive surgery or biopsies. Even after a definitive diagnosis is obtained, the prognosis is generally based on subjective factors.
In certain diseases, such as metabolic disease, the methods by which an objective diagnosis may be made are often cumbersome, time-consuming and costly. For example, the primary method for diagnosing type 2 diabetes is the fasting plasma glucose test, which assesses blood sugar levels in plasma. This test requires the patient to fast for 8-14 hours, and often requires multiple blood draws over a period of hours to days. In addition, although the fasting plasma glucose test is useful in diagnosing the presence of type 2 diabetes, the test is very limited in its ability to provide a disease prognosis.
In medicine, there is a constant search for less invasive, less physically taxing, and more accurate ways to diagnose and treat diseases or conditions. As a greater understanding of biological processes, and the biochemistry associated with these processes, unfolds, certain theories have evolved about which compositions might be identified as markers or indicators for certain diseases or conditions. Proteases and peptidases, as a class, have been investigated for their utility in diagnosis and as targets for treating patients.
By way of general background, proteases/petpidases are typically classified by a number of criteria, such as site of action, substrate preference, and mechanism. For example, aminopeptidases act preferentially at the N-terminal residues of a polypeptide, carboxypeptidases act preferentially at the C-terminus, and endopeptidases act at sites between these two termini.
Dipeptidyl peptidases (DPPs) are peptidases that specifically cleave a dipeptide unit, i.e., a two amino acid unit, from their specific substrates. There are a number of different DPPs, and substrate preference is frequently expressed in terms of the amino acid residue immediately N-terminal to the cleavage site. For example, DPP-I (IUBMB Enzyme Nomenclature EC.3.4.14.1) is a lysosomal cysteine-type peptidase that releases an N-terminal dipeptide, Xaa-Yaa-|-Zaa- except when Xaa is Arg or Lys, or Yaa or Zaa is Pro. DPP-II (IUBMB Enzyme Nomenclature EC.3.4.14.2) is a lysosomal serine-type peptidase that releases an N-terminal dipeptide, Xaa-Yaa-|-, preferentially when Yaa is Ala or Pro. DPP-III (IUBMB Enzyme Nomenclature EC.3.4.14.4) is a cytosolic peptidase that has a broad activity on peptides, although it is highly selective for Arg-Arg-Z, where Z is any amino acid, at pH 9.2. DPP-IV (IUBMB Enzyme Nomenclature EC.3.4.14.4) is a membrane-bound serine-type peptidase that releases an N-terminal dipeptide from Xaa-Yaa-|-Zaa-, preferentially when Yaa is Pro, provided Zaa is neither Pro nor hydroxyproline.
DPPs are involved in a wide range of physiologically important activities, and have been associated with regulation of the neurological system, endocrine system, immune system and digestive system. DPP activity has been demonstrated in numerous intracellular and extracellular functions such as protein degradation and enzyme activation.
With regard to the specific DPPs mentioned previously, DPP-IV has been widely studied, along with its attendant isoforms and isozymes or structural homologs, and those proteins that exhibit DPP-IV-like activity. Proteins which exhibit DPP-IV-like activity have been termed dipeptidyl peptidase IV activity and/or structure homologs, or “DASH”. DPP-IV is a type II membrane protein that is referred to by a number of names, including, but not limited to, DPP4, DP4, DAP-IV, FAP β adenosine deaminase complexing protein 2, adenosine deaminase binding protein (ADAbp), dipeptidyl aminopeptidase IV; Xaa-Pro-dipeptidyl-aminopeptidase; Gly-Pro naphthylamidase; postproline dipeptidyl aminopeptidase IV; lymphocyte antigen CD26; glycoprotein GP110; dipeptidyl peptidase IV; glycylproline aminopeptidase; glycylproline aminopeptidase; X-prolyl dipeptidyl aminopeptidase; pep X; leukocyte antigen CD26; glycylprolyl dipeptidylaminopeptidase; dipeptidyl-peptide hydrolase; glycylprolyl aminopeptidase; dipeptidyl-aminopeptidase IV; DPP IV/CD26; amino acyl-prolyl dipeptidyl aminopeptidase; T cell triggering molecule Tp103; X-PDAP. (Burgess et al., U.S. Pat. No. 7,169,926).
A number of DASH proteins have been reported, such as seprase, fibroblast activation protein a, DPP6, DPP8, DPP9, attractin, N-acetylated-α-linked-acidic dipeptidases I, II, and L, quiescent cell proline dipeptidase, thymus-specific serine protease and DPP IV-β (Busek et al., Int. J. Biochem. Cell Biol. 36:408-421 (2004)).
DPP-IV is constitutively expressed on epithelial and endothelial cells of a variety of different tissues, including intestine, liver, lung, kidney and placenta (Hartel et al., Histochemistry 89(2):151-161 (1988); Yaron and Naider, Critical Rev. Biochem. Mol. Biol. 28(1):31-81 (1993)). DPP-IV is expressed on circulating T-lymphocytes and has been shown to be synonymous with the cell-surface antigen, CD-26 (Sedo et al., Arthritis Res. Ther. 7:253-269 (2005)). In addition to a membrane-bound form, DPP-IV also exists in a soluble form, and DPP-IV activity can be found in body fluids such as blood plasma and synovial fluid (Sedo et al., Arthritis Res. Ther. 7:253-269 (2005); Gorrell, Clinical Sci. 108:277-292 (2005)).
DPP-IV is believed to play an important role in neuropeptide metabolism, T-cell activation, cell adhesion, digestion of proline containing peptides in the kidney and intestines, HIV infection and apoptosis, and regulation of tumorigenicity in certain melanoma cells (Mattem et al., Scand. J. Immunol. 33:737 (1991); Pethiyagoda et al., Clin. Exp. Metastasis 18(5):391-400 (2000)).
The natural substrates of DPP-IV include several chemokines, cytokines, neuropeptides, circulating hormones and bioactive peptides (Lambeir et al., J. Biol. Chem. 276(32):29839-29845 (2001)). A key regulatory role for DPP-IV, in the metabolism of peptide hormones and in amino acid transport, has been suggested. (Hildebrandt et al., Clin. Sci. (Lond.) 99(2):93-104 (2000)).
DPP-IV expression is increased in T-cells upon mitogenic or antigenic stimulation, suggesting a role in the immune system (Mattem et al., Scand. J. Immunol. 33:737 (1991)). Various other functions of T-lymphocytes such as cytokine production, IL-2 mediated cell proliferation and B-cell helper activity have also been shown to be dependent on DPP-IV activity (Schon et al., Scand. J. Immunol. 29:127 (1989)). In addition, DPP-IV appears to have a co-stimulatory function during T-cell activation and proliferation (von Bonin et al., Immunol. Rev. 161:43-53 (1998)).
DPP-IV is involved in other biological processes, including a membrane-anchoring function for the localization of the extracellular enzyme adenosine deaminase (ADA) (Franco et al., Immunol. Rev. 161: 27-42 (1998)) and participation in cell matrix adhesion by binding to collagen and fibronectin (Loster et al., Biochem. Biophys. Res. Commun. 217(1):341-348 (1995)).
DPP-IV is also believed to play a role in endocrine regulation and metabolic physiology. For example, DPP-IV cleaves the amino-terminal His-Ala dipeptide of glucagon like peptide-1 (GLP-1), generating a GLP-1 receptor antagonist, and thereby shortens the physiological response to GLP-1. DPP-IV has been implicated in the control of glucose metabolism because its substrates include the insulinotropic hormones GLP-1 and gastric inhibitory peptide (GIP), which are inactivated by removal of their two N-terminal amino acids. (Mannucci et al., Diabetologia 48:1168-1172 (2005)).
In addition to normal physiological function, DPPs have been studied for their role in disease states, including cancer, autoimmune disease, cardiovascular disease, metabolic disease and infectious disease.
For example, it has been suggested that DPP-IV is an adhesion molecule for lung-metastatic breast and prostate carcinoma cells (Johnson et al., J. Cell. Biol. 121:1423 (1993)). High DPP-IV activity has been found in tissue homogenates from patients with benign prostate hypertrophy and in prostatosomes (Vanhoof et al., Eur. J. Clin. Chem. Clin. Biochem. 30:333 (1992)).
High levels of DPP-IV expression have been found in human skin fibroblast cells from patients with the autoimmune diseases psoriasis, rheumatoid arthritis (RA) and lichen planus (Raynaud et al., J. Cell. Physiol. 151:378 (1992)).
DPP-IV has been associated with a number of metabolic diseases such as obesity and appetite regulation. For example, one of the more extensively studied DPP-IV-associated metabolic diseases is type 2 diabetes. Mannucci et al., defines and describes the relationships between chronic hyperglycemia and DDP-IV in diabetes. This research concludes that circulating DPP-IV activity directly correlates with the degree of hyperglycemia in type II diabetes.
Other studies discuss the relationship between DPP-IV and various hormones involved in the hormone cascade that regulates blood sugar levels. These studies conclude that DPP-IV degrades a hormone that is important for insulin secretion. Specifically, it has been suggested that DPP-IV degrades glucagon-like 1 peptide (GLP-1) which results in a decrease in insulin secretion and thus an increase in blood sugar. Based on this phenomenon, inhibitors of DPP-IV are being developed for the treatment of type II diabetes (Green et al., Diab. Vasc. Dis. Res. 3(3):159-165 (2006)).
DPP-IV is apparently essential for the penetration and infectivity of HIV-1 and HIV-2 viruses in CD4+ T-cells (Wakselman et al., J. Dermatol. Sci. 22:152-160 (2000)). Therefore, there is some suggestion that suppression of DPP-IV might suppress this mechanism as well.
Recently, some avenues of DPP research have focused on the manipulation of DPP levels as a means for developing treatments and therapies for the DPP-associated disease states and conditions. However, few treatments and therapies have resulted from this work to date.